Identification of natural genomic variants that determine cryptococcal pathogenicity Cryptococcus neoformans is a global pathogen responsible for hundreds of thousands of deaths yearly in HIV+ individuals and increasing morbidity in non-AIDS patient populations. Striking differences in virulence are observed among naturally occurring strains of this microbe, but no natural genomic variant responsible for this differential virulence has yet been reported. The goal of this research is to fill this gap in knowledge. Doing this will require overcoming the current challenges of insufficient strain diversity in individual strain collections, reli- ance on multi-locus sequence typing (MLST) to characterize genomes, and confounding host factors at the levels of the host genome and underlying morbidities. We hypothesize that by assessing the virulence of a diverse group of whole genome sequenced strains in a standardized mouse model, we will be able to computationally identify and experimentally validate natural variants that influence virulence. In this R21 application we propose to test this hypothesis with an initial set of clinical isolates. The range of analyses required to achieve our goal will be enabled by the synergistic efforts of two labs with complementary skills sets in computational and experimental biology and a history of productive collaboration on C. neoformans. In Aim 1 we will assemble whole genome sequences (WGS) and corresponding mouse infection data from diverse clinical isolates and progeny of C. neoformans genetic crosses. In Aim 2 we will perform genome-wide association studies (GWAS) and bulk segregant analysis (BSA), interpreted using our expertise in cryptococcal biology and gene regulation, to generate and prioritize hypotheses about which genomic variants influence virulence. In Aim 3 we will directly test a subset of high-priority hypotheses by genome engineering and virulence studies. The significance of this application lies in the impact of the pathogen on human health; the likely new insights into basic biology of cryptococcal gene regulation and protein function; and the potential for future application in terms of disease surveillance, patient stratification, and identification of new therapeutic targets. Innovative aspects include the use of diverse clinical isolates, bulk segregant analysis, a novel computational pipeline, and experimental validation of hypothesized causal variants. Together, these studies will provide a template for investigations of natural variants in C. neoformans and their role in virulence, generate significant resources for the research community, and potentially identify and validate causal variants that influence virulence. This exploratory proposal will additionally lay the groundwork for future studies of larger strain sets and follow-up by us and others in the directions of both fundamental biological understanding and potential application.